Arginine methylation in subunits of mammalian pre-mRNA cleavage factor I

Area of Science:

• Molecular Biology
• Biochemistry
• RNA Metabolism

Background:

• Mammalian cleavage factor I (CF I(m)) is crucial for messenger RNA (mRNA) precursor 3' end processing.
• Post-translational modifications, including arginine methylation, play significant roles in regulating protein function.
• Protein arginine methyltransferases (PRMTs) catalyze the transfer of methyl groups to arginine residues.

Purpose of the Study:

• To investigate post-translational modifications of CF I(m) subunits.
• To identify specific PRMTs involved in the methylation of CF I(m) components.
• To elucidate the sites and types of arginine methylation on CF I(m)68.

Main Methods:

• In vitro methylation assays using HeLa cell extracts.
• Fractionation of cell extracts and mass spectral analysis.
• Site-directed mutagenesis and in vitro methylation assays to confirm methylation sites.

Main Results:

• CF I(m)68 and nuclear poly(A) binding protein 1 (PABPN1) were identified as substrates for methylation.
• PRMT5, with cofactor WD45, was found to symmetrically dimethylate CF I(m)68, with pICln acting as a stimulatory factor.
• PRMT1 was identified as an enzyme responsible for asymmetric dimethylation of the GAR motif in CF I(m)68 and weak C-terminal methylation of CF I(m)59 and CF I(m)68.
• Methylation sites were exclusively identified within a conserved vertebrate GAR motif in CF I(m)68.

Conclusions:

• CF I(m)68 undergoes both symmetric and asymmetric dimethylation by PRMT5 and PRMT1, respectively.
• Methylation occurs within a conserved GAR motif, suggesting functional significance.
• Native protein substrates may possess determinants influencing specific PRMT activity.
• CF I(m) methylation might be involved in RNA export processes.